The prevalence of polio virus has largely been decreased by the use of Oral Polio Vaccine (OPV), based on live-attenuated Sabin polio strains. However, OPV has limitations for the post-eradication era. Therefore, development of Sabin-IPV plays an important role in the WHO polio eradication strategy. The use of attenuated Sabin instead of wild-type Salk polio strains will provide additional safety during vaccine production. Moreover, to prevent the emergence of circulating vaccine-derived polioviruses (cVDPVs), the use of OPV should be discontinued following polio eradication, and replaced by IPV. These cVDPVs are transmissible and can become neurovirulent (similar to wild polioviruses) resulting in vaccine associated paralytic poliomyelitis. Such strains can potentially re-seed the world with polioviruses and negate the eradication accomplishments.
IPV is delivered by intramuscular (IM) or deep subcutaneous (SC) injection. IPV is currently available either as a non-adjuvanted stand-alone formulation, or in various combinations, including DT-IPV (with diphtheria and tetanus toxoids) and hexavalentDTPHepB-Hib-IPV vaccines (additionally with pertussis, hepatitis B, and Haemophilus influenzae b. The currently acceptable standard dose of polio vaccines contains D antigens as 40 Units of inactivated poliovirus type 1 (Mahoney), 8 units of inactivated poliovirus type 2 (MEF-I) and 32 units of inactivated poliovirus type 3 (Saukett) (e.g. Infanrix-IPV™). Existing preparations of stand-alone IPV do not contain adjuvant.
Most experts agree that worldwide use of IPV is preferable because of its proven protective track-record and safety. However, when compared to OPV, the cost-prize for IPV is significantly higher. This is mainly due to requirements for: (i) more virus per dose; (ii) additional downstream processing (i.e. concentration, purification and inactivation), and the related QC-testing (iii) loss of antigen or poor recovery in downstream and iv) containment. Until now, the financial challenge has been a major drawback for IPV innovation and implementation in low and middle-income countries. The production costs of sIPV are currently estimated equivalent to that for IPV, which is about 20-fold more expensive than OPV. The future global demand for IPV following eradication of polioviruses could increase from the current level of 80 million doses to 450 million doses per year. Consequently, approaches to “stretch” supplies of IPV are likely to be required.
Reduced-dose efficacious vaccine formulations which provide protection against infection using a lower dose of IPV antigen are desirable in situations where the supply of conventional vaccine is insufficient to meet global needs or where the cost of manufacture of the conventional vaccine prevents the vaccine being sold at a price which is affordable for developing countries. Also the exposure to lower dose of IPV; compared to the existing marketed formulations could be more safer. Thus, various strategies to make IPV available at more affordable prices need to be evaluated.
In case of pandemic influenza vaccines the use of adjuvants has permitted dose reduction, increased the availability and reduced cost of the vaccine. Therefore, it has been speculated that an adjuvanted vaccine formulation of sIPV would reduce cost and also increase the number of available sIPV doses worldwide.
Globally different research groups have been evaluating dose sparing for vaccines (Influenza vaccines in particular) by employing several adjuvants namely Alum, Emulsion, TLR-agonists (MPL, CpG, poly-IC, imiquimod), dmLT, 1,25-dihydroxyvitamin D3, CAF01, poly [di (carboxylatophenoxy)-phosphazene] (PCPP) and Venezuelan equine encephalitis (VEE) replicon particles. Most of the adjuvant types being studied have encountered following hurdles i) Unknown safety or classified as toxic by regulatory agencies ii) having limitations regards to route of administration iii) lacking manufacturing reproducibility iv) stability of adjuvant.
Emulsion adjuvants (MF-59, AS03, AF3) have been previously reported to provide a strong dose-reduction effect (>30 fold) for Influenza and Hepatitis B vaccines. These adjuvants work by forming a depot at the site of injection, enabling the meted release of antigenic material and the stimulation of antibody producing plasma cells. However, these adjuvants have been deemed too toxic for widespread human prophylactic vaccine use and are usually reserved for those severe and/or terminal conditions such as cancer where there is a higher tolerance of side-effects.
Further, Aluminum salts have been considered safe, are already being used in combination vaccines containing sIPV, have the lowest development hurdles and are inexpensive to manufacture. However aluminium adjuvants are not known for permitting significant dose-reduction.
One of the most critical steps in the production of vaccines against pathogens, in particular viral vaccines, is viral inactivation. In the case of virus inactivation, formalin is the most frequently used inactivating agent in the manufacture of vaccines. Formaldehyde inactivates a virus by irreversibly cross-linking primary amine groups in surface proteins with other nearby nitrogen atoms in protein or DNA through a —CH2-linkage. A potential problem with using formalin for viral inactivation is that this involves a series of chemical reactions that produce reactive products that can induce cross-linking of viral proteins and aggregation of virus particles. This could hamper the inactivating efficiency of the formalin and could also result in the partial destruction of the immunogenicity of the antigen in vaccine. Accordingly, it has been reported previously that formalin inactivation of polioviruses could affect the viral immunogenicity as well as antigenicity. Refer Morag Ferguson et al Journal of General Virology (1993), 74, 685-690. Most importantly, previously disclosed formaldehyde inactivation methods were particularly carried out in presence of phosphate buffer wherein significant D-antigen losses were observed along with epitope modification for Sabin Type I/II/III (D-antigen recovery post inactivation: 22% for sabin type I, 15% for sabin type II, 25% for sabin type III), thereby failing to preserve the epitopic conformation. It is therefore possible that antibodies produced by recipients of formalin-inactivated polioviruses (in presence of phosphate buffer) may not contribute to the protective immune response.
By combining formalin and UV-inactivation, scientists tried to overcome the limitations of isolated UV-inactivation or formalin-inactivation, respectively, when inactivating the particularly resilient poliovirus. See, e.g., McLean, et al., “Experiences in the Production of Poliovirus Vaccines,” Prog. Med. Virol., vol 1, pp. 122-164 (1958.) Taylor et al. (J. Immunol. (1957) 79:265-75) describe the inactivation of poliomyelitis virus with a formalin and ultraviolet combination. Molner et al. (Am. J. Pub. Health (1958) 48:590-8) describe the formation of a measurable level of circulating antibodies in the blood of subjects vaccinated with ultraviolet-formalin inactivated poliomyelitis vaccine. Truffelli et al. (Appl. Microbiol. (1967) 15:516-27) report on the inactivation of Adenovirus and Simian Virus 40 Tumorigenicty in hamsters by a three stage inactivation process consisting of formalin, UV light and β-propiolactone (BPL). Miyamae (Microbiol. Immunol. (1986) 30:213-23) describes the preparation of immunogens of Sendai virus by a treatment with UV rays and formalin. However previously discussed promising alternatives for formaldehyde like β-propiolactone (BPL) have been reported to produce an immune complex-reaction when combined with other components of the rabies vaccine. Additionally, it has been shown to produce squamous cell carcinomas, lymphomas and hepatomas in mice.
It is therefore particularly desirable to employ favorable formaldehyde inactivation conditions that maintain the structural integrity of antigenic structures of Sabin strains as well as utilize safe and cost-effective adjuvants that can result in significantly dose reduced (i.e. 8 to 10 fold) sIPV (Sabin IPV) vaccine compositions thereby reducing cost of manufacture, increasing vaccine supplies and making vaccines affordable for developing countries.
The present inventors have surprisingly found that D-antigen losses post-formaldehyde inactivation could be due to presence of phosphate buffer that unexpectedly causes undesirable aggregation of polio viruses. The instant invention provides an improved process of formaldehyde inactivation in presence of TRIS buffer thereby ensuring minimal epitopic modifications and subsequently minimizing D-antigen losses. Subsequently significantly dose reduced Sabin IPV vaccine compositions with atleast 8 fold dose reduction for Sabin Type I and 3 fold dose reduction for Sabin Type III can be obtained.